收稿日期: 2017-04-03
网络出版日期: 2017-06-30
基金资助
国家自然科学基金资助项目(81370280, 81570332)
Human embryonic stem cell-derived cardiomyocytes model establishment and systemic identification scheme
Received date: 2017-04-03
Online published: 2017-06-30
心肌细胞是研究心血管疾病的重要工具之一, 但是人类心肌细胞较难获得和培养. 为人类胚胎干细胞诱导分化成心肌细胞提供一个有用的实验方法和鉴定方案. 人胚胎干细胞以其多向分化的特性为体外研究提供了细胞资源. 在人胚胎干细胞诱导分化为心肌细胞的过程中, 通过荧光定量聚合酶链式反应(polymerase chain reaction, PCR) 检测发现, 在分化过程中干细胞标记物Cripto, Dnmt3b, Wnt3, KIF4, Oct4, SOX2 和Nanog 表达下降, 心肌特异性结构蛋白cTnT 和α-actinin 以及心脏前体细胞分化标记物Nkx2.5 表达上升. 分化完成后用免疫荧光检测心肌特异性结构蛋白TnnT2 和α-actinin, 通过分析TnnT2 阳性细胞的比例,α-actinin阳性细胞的比例, 以及TnnT2和α-actinin双阳性细胞的比例发现, 在所提出的胚胎干细胞诱导分化体系中, 三者比例分别为90.80%, 91.00%, 90.91%, 表明在此诱导分化条件下
人胚胎干细胞可成功分化成为心肌细胞. 成功建立了人胚胎干细胞来源的心肌细胞模型以及基于标记物荧光定量PCR 及免疫荧光系统检测的鉴定方案, 为未来心血管疾病的基础研究及心脏毒性药物检测奠定了一定的基础.
陈欣欣, 刘珠媛, 顾寰宇, 周蕾 . 人类胚胎干细胞来源的心肌细胞模型的建立及系统鉴定方案[J]. 上海大学学报(自然科学版), 2017 , 23(3) : 378 -386 . DOI: 10.12066/j.issn.1007-2861.1939
Cardiomyocytes is useful in the study of many cardiovascular diseases, while human cardiomyocytes are difficult to obtain and culture. With multi-directional differentiation properties, human embryonic stem cells can provide cell resources in vitro. This study aims to establish a useful protocol to induce the differentiation of human embryonic stem cells into cardiomyocytes, and observe identification of human cardiomyocytes. Expression of stem cell markers including Cripto, Dnmt3b, Wnt3, KIF4, Oct4, SOX2 and Nanog decrease during differentiation while cardiac progenitor cell marker Nkx2.5 and cardiomyocytes specific markers TnnT2 and a-actinin increase. Ratios of TnnT2 positive cells, α-actinin positive cells, TnnT2 and α-actinin double positive cells are 90.80%, 91.00%, and 90.91%. These results indicate that human embryonic stem cells can be efficiently induced into cardiomyocytes in the proposed protocol. In conclusion, differentiation of human embryonic stem cells into cardiomyocytes has been successfully induced and an identification scheme has been established, and markers have been detected through quantitative PCR and immunofluorescence stainings. The protocol will facilitate better understanding
of pathogenesis of cardiovascular diseases and enable better cardiotoxicity drug screening.
Key words： cardiomyocytes; cell differentiation; human embryonic stem cell
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